Unconventional Secretion of Park7 Requires Lysosomal Delivery via Chaperone-Mediated Autophagy and Specialized SNARE Complex

1. Research Background

The Parkinson’s disease-associated protein PARK7/DJ-1 (hereafter referred to as PARK7) is a multifunctional protein that plays critical roles in various pathological conditions including neurodegenerative diseases, cancer, and inflammation. Despite lacking a conventional N-terminal signal peptide sequence, PARK7 can be secreted extracellularly under stress conditions, with its secretion levels significantly elevated in the cerebrospinal fluid and blood of patients with multiple diseases. However, the specific mechanisms underlying PARK7’s unconventional secretion have long remained unclear.

Previous studies indicated that 6-hydroxydopamine (6-OHDA)-induced oxidative stress promotes PARK7 secretion through autophagy pathways. Yet key questions remained unresolved: 1. The molecular mechanism by which PARK7 is selectively translocated to the lysosomal lumen under oxidative stress 2. The composition and function of specialized SNARE (soluble N-ethylmaleimide-sensitive factor attachment protein receptor) complexes mediating PARK7 secretion

2. Paper Source

This study was conducted by a team led by Biplab Kumar Dash and Yasuomi Urano (corresponding author) from the Graduate School of Life and Medical Sciences at Doshisha University, Japan. The paper was published in Proceedings of the National Academy of Sciences (PNAS) Volume 122, Issue 19 (2025), DOI:10.1073/pnas.2414790122.

3. Research Process and Results

1. Validation of 6-OHDA-Induced Unconventional PARK7 Secretion

Experimental Design: - Used HeLa cell line treated with varying concentrations of 6-OHDA - Collected conditioned media after serum-free culture - Enriched secreted proteins via trichloroacetic acid precipitation, detected PARK7 secretion by Western blot

Key Findings: - Dose-dependent increase in PARK7 secretion with 6-OHDA (Fig.1B) - Brefeldin A (BFA) blockade of ER-Golgi pathway didn’t affect secretion (Fig.1D) - Ultracentrifugation showed PARK7 exclusively in soluble fraction (Supplementary Fig.S1A) - GW4869 inhibition of exosome release didn’t alter PARK7 secretion (Supplementary Fig.S1B)

Conclusion: 6-OHDA-induced PARK7 secretion is independent of conventional secretory pathways and exosome release.

2. Role of Autophagic Flux in PARK7 Secretion

Innovative Methods: - Tandem fluorescent-tagged LC3 (tfLC3) reporter system: Used GFP (acid-sensitive) and mRFP (acid-stable) to distinguish autophagosomes (yellow puncta) from autolysosomes (red puncta)

Key Experiments: - 6-OHDA treatment increased LC3-II conversion and SQSTM1/p62 degradation (Fig.2A) - Bafilomycin A1 (BafA1) and chloroquine (CQ) treatments further enhanced LC3-II accumulation (Fig.2B) - tfLC3 showed 6-OHDA increased both autophagosomes and autolysosomes (Fig.2C)

Mechanistic Validation: - ULK1 inhibitor MRT68921 and FIP200 knockout cells suppressed secretion (Fig.3A-C) - Rapamycin-induced autophagy enhanced secretion (Fig.3D) - Antioxidant N-acetylcysteine (NAC) inhibited 6-OHDA-induced secretion (Fig.3E-F)

3. Critical Role of Lysosomal Function

Technical Breakthroughs: - Subcellular fractionation with lysosomal marker LAMP1 detection (Fig.4C) - Immunofluorescence colocalization analysis of PARK7 and LAMP2

Major Discoveries: - Lysosomal inhibitors (CQ+NH4Cl) caused intracellular PARK7 accumulation (Fig.4A) - STX17 (essential for autophagosome-lysosome fusion) knockout significantly inhibited secretion (Fig.4D-E) - However, SNARE partners SNAP29/47 knockdown didn’t affect secretion (Fig.5)

4. Identification of Specialized SNARE Complex

Methodological Innovations: - AlphaFold2 multimer predicted protein complex structures (Supplementary Fig.S5F-G) - Co-immunoprecipitation validated SNARE interactions (Fig.7G)

Key Results: - Sec22b (R-SNARE) knockdown inhibited secretion (Fig.7B) - STX3/4 (plasma membrane Qa-SNARE) double knockout completely blocked secretion (Fig.7C) - Vti1b (Qb-SNARE) and STX8 (Qc-SNARE) knockdown significantly suppressed secretion (Fig.7E-F) - 6-OHDA promoted SNARE complex formation (Fig.7G)

5. CMA-Mediated PARK7 Translocation Mechanism

Novel Findings: - PARK7 contains four conserved KFERQ-like motifs (Supplementary Fig.S6D) - Site-directed mutagenesis revealed critical roles of residues ⁹¹⁄₉₂, ⁹⁴⁄₉₅, and ⁴⁴⁄₄₅ (Fig.8B-C) - Hspa8 and LAMP2 knockdown inhibited secretion (Fig.8D) - Crosslinking experiments showed secreted PARK7 was predominantly monomeric (Fig.8F)

4. Research Conclusions and Significance

This study is the first to demonstrate: 1. Molecular Mechanism: 6-OHDA induces PARK7 monomerization via oxidative stress, exposing KFERQ-like motifs for Hspa8-LAMP2-mediated CMA translocation to lysosomal lumen 2. Secretory Pathway: Specialized SNARE complex (STX3/4-Vti1b-STX8-Sec22b) mediates secretory autolysosome-plasma membrane fusion 3. Conceptual Innovation: Proposes new paradigm of “secretory autolysosome-mediated secretion”

Scientific Value: - Provides mechanistic explanation for aberrant protein secretion in neurodegenerative diseases - Reveals novel CMA function in protein secretion - Identifies molecular targets for therapeutic modulation of PARK7 secretion

5. Research Highlights

1. Methodological Innovations: Integrated tfLC3 live-cell imaging, AlphaFold2 structure prediction, and crosslinking mass spectrometry
2. Mechanistic Breakthrough: First to link CMA pathway with SNARE-mediated membrane fusion
3. Clinical Implications: Offers new perspectives for biomarker development and treatment of Parkinson’s and other neurodegenerative diseases

6. Additional Findings

• TMED10 knockout unexpectedly increased PARK7 secretion (Fig.8A), suggesting unknown regulatory pathways
• Lysosomal membrane permeabilization (LMP) assays confirmed 6-OHDA doesn’t compromise lysosomal integrity (Fig.6)
• Proteasome inhibition didn’t affect secretion (Supplementary Fig.S4), confirming pathway specificity